This invention is generally in the field of methods for the treatment and prevention of inflammatory responses involving GMP-140 binding reactions, particularly aggregation of platelets and leukocytes.
The U.S. Government has rights in this invention by virtue of grants from the National Heart, Lung and Blood Institute.
The adherence of leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems. Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion. Although a class of inducible leukocyte receptors, the CD11-CD18 molecules, are thought to have some role in adherence to endothelium, mechanisms of equal or even greater importance for leukocyte adherence appear to be due to inducible changes in the endothelium itself. Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for neutrophils within two to ten minutes, while endothelium exposed to cytokines such as tumor necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelial-dependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, although direct demonstration of the adhesive role of PAF has been more difficult to demonstrate and additional mechanisms for the early adhesion have not been examined. The later cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an endothelial cell receptor, ELAM-1, that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface.
Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro. The interaction of platelets with monocytes may be mediated in part by the binding of thrombospondin to platelets and monocytes, although other mechanisms have not been excluded. The mechanisms for the binding of neutrophils to activated platelets are not well understood, except that it is known that divalent cations are required. The in vivo significance of platelet-leukocyte interactions has not been studied carefully. However, in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion.
The complement proteins collectively play a leading role in the immune system, both in the identification and in the removal of foreign substances and immune complexes, as reviewed by Muller-Eberhard, H. J., Ann. Rev. Biochem. 57:321-347 (1988). Central to the complement system are the C3 and C4 proteins, which when activated covalently attach to nearby targets, marking them for clearance. In order to help control this process, a remarkable family of soluble and membrane-bound regulatory proteins has evolved, each of which interacts with activated C3 and/or C4 derivatives. This group of proteins constitutes the regulators of complement activation (RCA) gene family, reviewed by D. Hourcade, et al., Advances in Immunology, Volume 45, F. E. Dixon, ed., 381-416 (New York, Academic Press 1989), which are clustered at the q32 region of chromosome 1.
The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes. For example, the Mac-1 receptor on leukocytes, a member of the CD11-CD18 group, mediates phagocytosis and serves as a receptor for the degradation product of complement C3bi, is involved in one pathway of adherence of leukocytes to endothelium, mediates granulocyte aggregation, and binds coagulation Factor X.
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. An example is GMP-140 (granule membrane protein 140), an integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
GMP-140 was first purified from human platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984). Monoclonal and polyclonal antibodies to GMP-140 were also prepared, as reported by McEver and Martin (1984) and P. E. Stenberg, et al., J.Cell Biol. 101:80-886 (1985). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 (1987). In endothelial cells, GMP-140 is found in storage granules known as the Weibel-Palade bodies.
When platelet or endothelial cells are activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound GMP-140 is presented within seconds on the cell surface. While the rapid redistribution of GMP-140 to the surface of platelets and endothelial cells as a result of activation suggests that this glycoprotein could play an important role at sites of inflammation or vascular disruption, locations where both cell types would be activated, the function of this protein has not previously been determined.
It is therefore an object of the present invention to elucidate and characterize the structure and function of GMP-140.
It is another object of the present invention to provide a surface bound ligand for GMP-140.
It is a further object of the present invention to provide a method and means for using GMP-140 and the GMP-140 ligand in modulating the immune response and the hemostatic pathway, particularly in abnormal responses involving adherence of leukocytes to platelets and endothelium.
It is a still further object of the present invention to provide a method and means for screening for defects in the immune response, especially defects in GMP-140 or in ligands for GMP-140 or related proteins.